Process for the preparation of an extract with carotenoids, UV absorption, antibacterial and pH indicating properties from a deep-sea bacterium

ABSTRACT

Accordingly the present invention provides a process for the preparation an alcoholic extract with Carotenoids, UV absorption, antibacterial and pH indicating properties from a deep-sea bacterium which comprises a method for growing the cells in a medium with salinity ranging from 1.5 to 3% for 3-4 days at 28 +/−2° C. and harvesting them to prepare an extract which shows the properties of carotenoids (yellow/orange coloration), UV absorption, antibacterial and pH indicator properties.

FIELD OF THE INVENTION

[0001] The present investigation relates to a process for thepreparation of an extract with Carotenoids, UV absorption, antibacterialand pH indicating from a deep-sea bacterium for applications in food andcosmetic industries. The extract could be used as food and feedadditives (colorant), food and feed preservatives and radio protective Isunscreen compound in cosmetics.

BACKGROUND AND PRIOR ART REFERENCES

[0002] A number of bacteria are known to produce several secondarymetabolites like pigments, toxins, growth promoting compounds andantibiotics (Bushell M E, 1982, Microbial aspects of the discovery ofnovel secondary metabolites, Top. Enzyme-Ferment Biotech no 1 6, 32-67).These bacteria have an added advantage in that they are easilyreplenishable as their generation time is much shorter than othermicrobes and higher organisms. Besides, their products are eco-friendlyand can be used either in their native form or with minor modifications.

[0003] The synthetic pigments and dyes have proved to be more harmfulthan natural products. Some of the synthetic colours are either bannedor the permissible concentrations drastically reduced. In the Governmentof India notification an amendment to the Prevention of FoodAdulteration Act in March 1993 banned the use of permitted colours (TheNavhind Times dated Aug. 4, 1998) European Union is planning to banabout 300 textile dyes containing chemicals known to increase the riskof cancer. Dyes containing significant levels of 22 of the so-calledaromatic amines are known to be carcinogenic (The Navhind Times, datedOct. 4, 1999). The planned legislation would phase out dyes that areused in textile, leather goods, seat covers, gloves. In short anymaterial having even temporary contact with the skin, public'sinclination towards bio pigments and colours is strong.

[0004] Pigments are known to be the most important secondary metabolitesof organisms for various applications. Even though pigments can beproduced by chemical synthesis there has been increasing demand andrenewed interest for natural pigments. This is essentially due to thepublic awareness of the superiority and safety of natural products overthe synthetic ones, especially in the food industry.

[0005] Reference may be made to the natural colorants like red betalinpigment from plant, Beta vulgaris. (Smith M A L, Dustin I, Leathers R,Zryd J p 1992. Development of automated vision techniques for immediateanalysis and control of betalin producing cultures. Hortiscience 27 (6),572) or bright colour from the bryozoan animal, Bugula dentata(Matsunaga S, Fusetani N, Hashimoto K, 1986. Bioactive marinemetabolites. VIII Isolation of an antimicrobial blue pigment from thebryozoan Bugula dentata Experientia 42(1), 84). However, use of suchpigments from higher organisms has got certain limitation. For examplethe major drawbacks are that higher plants and animals have a longergeneration time and the resources are limited. Though tissue culture hasevolved as an alternate method to produce the cells for pigmentextraction, the techniques are not always simple and have limitedapplication. On the other hand, microbes, especially bacteria, can beexploited more advantageously for reasons mentioned above.

[0006] Among the pigments yellow-orange to red pigments, mostlybelonging to the carotenoid group has been popular for variousapplications. For example, B carotene is a valuable metabolite and is ingreat demand in the food industry and Aquaculture (Michel P J, DujardinE, Sironval C1995. Growth of Dunalliella bardawil under carotogenicconditions. J. Mar. Biotech no1 2 (2), 101-104). Carotenoids possessingcarbonyl groups inhibit lipid peroxidation better than those lackingcarbonyl groups (Michel P J,Dujardin E, Sironval C,1995. Growth ofDunalliella bardawil under carotogenic conditions. J. Mar. Biotechnol 2(2), 101-104). Despite the enormous economic potential of carotenoids,only a few of the microbes have been exploited commercially. Zeaxanthin,a carotenoid, from a bacterium (Alteromonas sp) is used in the foodindustry essentially for imparting colour to the product (Japanesepatent No. JP-5049497). The same compound from another bacteriumFlavobacterium sp (Nelis H J, De Leenheer A P 1991. Microbial sources ofcarotenoids pigments used in foods and feeds. J of Appl Bacterial 70,181-191. U.S. Pat. Nos. 841,967, 3,951,742, 3,951,743, 4,026,949) hasalso been reported. A formulation of zeaxanthin from Flavobacteriummultivorans is also claimed to prevent degeneration of macula in theiris of the eye (U.S. Pat. No. 5,827,652). Astaxanthin, anothercarotenoid from the yeast, Phaffia rhodozyme (Nelis H J, De Leenheer A P1991. Microbial sources of carotenoid pigments used in foods and feeds.J of Appl Bacteriol 70, 181-191) has been reported to be useful asgrowth promoters Another group of pigments like menaquinones extractedfrom bacteria have also been used for various applications. A processhas been claimed for the preparation of menaquinone-containing substancefrom Bacillus subtilis in food and drink preparations for osteoporosistherapy (JP-1 1032787). Pigments from Brevibacterium species have beenused in the dairy products especially for ripening and flavouring cheese(Reyser ET, Maisnier-Patin S, Gratadoux J J & Richard J 1994. Isolationand identification of cheese smear bacteria inhibitory to Listeria spp.Int. J Food Microbiol. 21, 237-246) and production of menatetrone(JP-63267283; JP-61173792; EP-202613).

[0007] Japanese Patent No. 95-05169, titled: “Cosmetic containingfermented Streptomyces sp.” describes “a new cosmetic preparationcontains fermentation broth from Streptomyces sp.

[0008] G172 (FERM P-13630), grown in culture medium at 25-30 deg for 4-7days. The fermentation broth is extracted and purified to form a clearsolution. The cosmetic is useful for whitening skin, or as a sunscreen.In an example G172 was grown at 25 deg for 5 days, centrifuged andmaintained at −20 deg overnight to give a precipitate. The precipitatewas filtered, and a skin lotion was prepared from the fermentation broth(10 wt. %), glycerol (5%), polyoxyethylene sorbitan monolaurate (1.5%),ethanol (10%), fragrance, antioxidant, antiseptic, pigment and purifiedwater (6pp)”.

[0009] Japanese Patent No.JP07010736; 13.01.95; 128:66319 Coupland,Keith; Packer, Clarie Elizabeth (Croda International PLC; Coupland,Keith; Packer, Claire Elizabeth, UK) titled: “Sunscreen compositionscomprising stearidonic acid and derivatives in combination with a UVblocking and/or absorbing material”, describes “A sunscreen compositioncomprising a stearidonic acid, or a physiol. deriv. thereof, incombination with a UV blocking and/or UV absorbing material, is claimed.Also stearidonic acid may be used to treat inflammation caused byexposure to UV radiation, by exposure to sunlight or by burns. Thus, 10kg of the seeds of Echium plantagineum were crushed and the oil wasextracted with 15 L of petroleum ether. The petroleum ether extract wasevaporated to yield 1741 g of golden yellow oil. The oil was convertedto the corresponding fatty acid Me esters and used in sunscreens. Asunscreen oil was prepared containing Bu methoxydibenzoyl methane 2.0,octyl methoxy-cinnamate 7.5, benzophenone-3 4 5, PPG-2 myristyl etherpropionate 10.0, above oil 2.0-10.0, perfumes, preservatives, andcaprylic/capric triglycerides q.s. 100%.

[0010] Sophie Maisnier-patin and J. Richard, Station de RecherchesLaitieres, Institut National de la Recherche Agronomique, Jouy-en-Joses,France, (Applied and Environmental Microbiology, May 1995, p.1847-1852);titled: Activity and Purification of Linenscin OC2, an AntibacterialSubstance Produced by Brevibacterium linens OC2, an Orange CheeseCoryneform Bacterium, describes: An orange cheese coryneform bacteriumisolated from the surface of Gruyere of Comte and identified asBrevibacterium linens produces an antimicrobial substance designatedlinenscin OC2. This compound inhibits gram-positive food-borne pathogensincluding Staphylococcus aureus and Listeria monocytogenes but is notactive against gram-negative bacteria”.

[0011] Japanese Patent No.99-06310; titled: “A process for preparationof menaquinone-7 containing substance and food and drink preparations”describes: “A process is claimed for the preparation of menaquinone-7(I) for use in food and drink preparations. (I) is produced by culturingBacillus subtilis MR-141 (FERM P-14692), S-2 (IFO 14898, FERM BP-2528),ATCC 162 (FERM P-1 1052), F-2-01 (FERM P-9082) or their mutants in aliquid nutrient media at 30-40 deg, pH 6-8 for 2-4 days. The cells areremoved by filtration and centrifuged. Both the cell pellet and thesupernatant are dehydrated (e.g. by spray drying, lyophilization anddrying in vacuo) in the absence of an organic solvent. The product isused for the prevention and treatment of osteoporosis (no clinical datadisclosed) and is included in dried foods at 0.1-10% and drinkpreparations at 0.1-5%”.

[0012] Most of the commercially available UV-blocking compounds in skincream (sunscreen) are synthetic and the search for natural compoundswith equal or greater efficiency is becoming more significant because ofthe consumer's preference for natural products.

[0013] The UV-absorbing properties of either the organisms or theextract have been extensively studied in higher plants, corals,cyanobacteria and other phytoplankton. Reference may be made to an UVabsorbing (310 nm) compound that has been characterised from stem, barkand roots of mangrove plant, Heritiera littoralis (Bandaranayake W M1994. phyto-chemical constituents and pigments in mangrove species andmangal associates of Northern Australia. Aust Inst Mar Sci RepTownsville, Old Australia AIMS 19, 28pp). The hyperoxic tissues of coralreefs also produce UV absorbing mycosporine like compounds (Dunlap W CShick J M 1998. Ultraviolet radiation absorbing mycosporine-like aminoacids in coral reef organisms: A biochemical and environmentalperspective. J.Phycol. 34(3), 418-430). The induction and protectiverole of the UV-absorbing compounds such as mycosporine-like amino acids(MAAs) have been noted even in Florideophyceae (Franklin L A,Yakovleva.I, Karsten U, Luenig, K 1999. Synthesis of mycoporine-likeaminoacids in Chondrus crispus (Florideophyceae) and the consequencesfor sensitivity to ultraviolet B radiation. J.Phycol. 35,682-693)Certain algae like Dunaliella are also known to possess intensecarotenoid pigments to protect themselves against intense solarradiation. Some algae have other type of UV-absorbing (sunscreen)pigments like scytonemin (Proteau ,P. J., Gerwick, W. H. Garcia-PichelF. Castenholz 1993. The structure of scytonemin an ultraviolet sunscreenpigment from the sheath of cyanobacteria. Experientia 49, 825-829).These UV absorbing compounds are also known to be produced underphotoinductive conditions and are dependant on temporal factors (HannachG, Sigleo A C 1998. Photoinduction of UV absorbing compounds in sixspecies of marine phytoplankton. Mar. Ecol. Prog Ser 174; 207-222).Though a number of papers have been published on the ultravioletabsorbing/screening substances in cyanobacteria, phytoplankton andmacroalgae and their role in mitigating ultraviolet toxicity (Sinha R P,Klisch M, Groeniger A, Haeder D P. 1998. Ultraviolet absorbing/screeningsubstances in cyanobacteria, phytoplankton and macroalgae. J PhotochemPhotobiol B 47 J (2-3), 83-94) there have been only few publications onthis aspect of bacteria (Arai T. Nishijima M, Adachi K Sano H 1992.Isolation and structure of a UV absorbing substance from the marinebacterium Micrococcus sp AK 334. Marine Biotech Inst Rep. Pp88-94Japan). Sunscreen compositions comprising natural products of marinehydroid and derivatives thereof have been patented as usefulsunscreening agents (lindquist, N. l. 1998 U.S. Pat. No. 5,705,146). Asunscreen/radioprotective compound has also been patented from fungusAspergillus versicolor FK17 95-03294 (JP-06329576). Though bacteriapossess a number of pigments that are supposedly photo protective only afew have been used for extracting UV A and B absorbing components. Afat-soluble UV absorbing compound F-1547 from Paracoccus sp has beenpatented (JP-1 1269175). A process for producing UV absorbingmycosporine-like aminoacids (MAA) from Micrococcus sp has also beendeveloped (JP-06062878-A).

[0014] Antibacterial activity is widespread in microorganisms especiallyin those belonging to streptomycete group and many compounds from thisgroup have been discovered and the processes and products have beenpatented. Ditrisaburicins A, B, and C from Streptomyces cyaneus areactive against Gram-positive bacteria (Microb Chem. Res. Found.EP-110155) For example CV 1 which is an antibacterial compound fromStreptomyces sp is also used for sterilising instruments in hospitals(JP-J62000286). AAD-609 antibiotics produced by the fungusKibdelosporangium aridum ATCC 3922 are inhibitory to Gram-positivebacteria (EP-218416). Narrow spectrum bactriocins from bacteria that areactive against closely related species is common. Bacteriocins havewide-ranging applications in the food industry as preservatives (Eckner,K. F. 1992. Bacteriocins and Food Application. Dairy Food and Environ,Sanitation 12 (40), 204-209). Linenscin OC₂ is an antibacterialsubstance produced by the orange cheese coryneform bacteriumBrevibacterium linens OC₂. It is bacteriolytic to Listeria innocua.(Boucabeille C. Menginlecreulx D. Henckes G.Simonet JM, Vanheijenoort J.1997. Antibacterial and haemolytic activities of linocin OC₂ ahydrophobic substance produced by Brevibacterium linens OC₂ . FEMSMicrobiology Letters 153 (2), 295-301). The compound inhibitsGram-positive food-borne pathogen including Staphylococcus aureus andListeria monocyclogenes but is not active against Gram-negativebacteria. A process for the production of antibacterial compounddifficidin and its derivative is described where oxydifficidin isproduced in large amounts compared with the other derivatives (U.S. Pat.No. 4,545,991; Zimmerman S b, Schwartz C D, Monaghan R L, Pelak B A,Gillifillan E C 1987. Difficidin and Oxydifficidin; a novelbroad-spectrum antibacterial antibiotics produced by Bacillus subtilispreparation and characterisation. J. Antibiot, 40 (12), 1677-1681.)These compounds have been shown to be active against Klebsiellapneumonie. Antibacterial compounds from bacteria, which are activeagainst both Gram-positive and Gram-negative groups, are uncommon.

[0015] Some of the pigments in bacteria are also associated withantimicrobial activity. The use of antibiotic pigment violcaceinisolated from Janthinobacterium lividum ( J P-10113169 ) orChromobacterium violaceum and Janthinobacterium (JP-06253864) has beenpatented. Pigments with antimicrobial properties have been isolated evenfrom photosynthetic bacteria, Chromatium purpuratum (Burgess J G,Miyashita H, Sudo H, Matsunaga T 1991. Antibiotic production by marinephotosynthetic bacterium Chromatium purpuratum NKPB 031704: localisationof the activity to the chromatophores FEMS Microbiol lett. 84 (3),301-06). Cultures of aerobic marine bacterium Alteromonas rubra producered pigments with pH indicator properties and a compound showingantibiotic activity against a variety of bacteria (Laatsch H and ThomsonR H 1983. A revised structure for cycloprodigiosin.

[0016] Tetrahedron Lett. 24 (26), 2701-04).

[0017] Though there have been reports on the occurrence of pigmentsalone or pigments with antibacterial activity in some of the microbesand sun screen compounds from yet others, there is no report availableon the presence of all three in a single extract of a single bacterialspecies.

OBJECTS OF THE INVENTION

[0018] The main object of the present invention is to provide analcoholic extract of novel bacterium isolated from deep-sea of coastalzone of Arabian Sea, Goa, India.

[0019] Another objects of the present investigation are to provide aprocess for the preparation of an alcoholic extract, having Carotenoidsproperty, UV absorption, antibacterial and pH indicating properties.

[0020] Still another object of the invention is to use the extract froma deep-sea bacterium in food and cosmetics, which obviates the drawbacksas detailed above.

SUMMARY OF THE INVENTION

[0021] Accordingly the present invention provides a process for thepreparation an alcoholic extract with Carotenoids, UV absorption,antibacterial and pH indicating properties from a deep-sea bacteriumwhich comprises a method for growing the cells in a medium with salinityranging from 1.5 to 3% for 3-4 days at 28+/−2° C. and harvesting them toprepare an extract which shows the properties of carotenoids(yellow/orange coloration), UV absorption, antibacterial and pHindicator properties.

DETAILED DESCRIPTION OF THE INVENTION

[0022] Accordingly, the present invention provides an novel deep-seabacterium deposited with National Institute of Oceanography, Goa, India,having an accession no NIOCC isolate #222, and being deposited withInternational depository . . . having accession no . . . which has asimilar properties to known Brevibacterium casei isolated from the deepsea at 5000 m depth waters of the Indian Ocean.

[0023] In one embodiment of the invention, the novel bacterium is abaroduric (pressure tolerant) one i.e. it is capable of growing both at500 atm and at 1 atm pressure and the petroleum ether fraction of thebacterium when scanned in an UV visible spectrometer showscharacteristic peaks at 448 nm with shoulders at 430 and 470 nm, whichis similar to the caratenoid compound.

[0024] In another embodiment of the invention, the alcoholic extract ofthe said bacterium having caroteniods, UV absorption, anti bacterial, pHindicating properties.

[0025] In still another embodiment, the extract of the bacterium is usedin many industrial applications, such as a food and beverages additiveand food additive colour cum preservative.

[0026] The invention also describes a process for culturing the marineisolate for consistent cell biomass. Further, it elaborates the processof harvesting cells for bioactive pigments and associated radioprotective constituents. This is the first time that a single extract ofa baroduric marine isolate is being exploited for 3 different uses. Thenative extract of the culture is a pigment complex, which is yellow toorange in colour, has UV absorbing property and is antibacterial againstsome of the Gram-positive and Gram-negative bacteria.

[0027] The present investigation undertaken by the inventors has led tothe preparation of single extract from a single bacterial isolate whichshows multiple properties of colour, UV absorption, antibacterialproperty against both Gram-positive and Gram-negative bacteria, and pHindicator property. As a food additive, the preparation would not onlyadd colour but also improve shelf life because of the antibacterialproperty. Further, it would also indicate deterioration by change incolour. This is the first time that a deep-sea isolate is used toprepare a single extract with multiple uses.

[0028] One more embodiment of the invention provides a process for thepreparation of alcoholic extract of deep-sea bacterium isolated from theIndian coastal zones of Arabian sea, said process comprising isolatingthe bacterium and growing the cells in a medium with salinity rangingfrom 1.5 to 3% for 3-4 days at 28±2° C., centrifuging and washing with1.5% NaCl, extracting with alcohol for 2-3 times and obtaining anextract which shows the properties of carotenoids (yellow/orangecolour), UV absorption, antibacterial and pH indicator. In anotherembodiment, the solvent used for extraction is an alcohol preferablymethanol. In still another embodiment, the extract is used as UV (A, B,C) absorbing compound. In still another embodiment, the extract inhibitsgrowth of Gram-positive and Gram-negative bacteria.

[0029] In yet another embodiment, the yellow methanolic extract showsreversible colour change, being pink under alkaline and yellow underneutral or acidic conditions and is used as a pH indicator.

[0030] The present invention provides a process for the preparation analcoholic extract with Carotenoids, UV absorption, antibacterial and pHindicating properties from a deep-sea bacterium for use in food andcosmetic industries which comprises a method for growing the cells in amedium with salinity ranging from 1.5 to 3% for 3-4 days 28+/−2° C. andharvesting them to prepare an extract which shows the properties ofcarotenoids (yellow/orange coloration), UV absorption, antibacterial andpH indicator properties.

[0031] In an embodiment of the present invention, the extract ofbacterial cells obtained from the deep-sea is has carotenoid property.

[0032] In another embodiment of the present invention, the extractpossesses UV absorbing property. In yet another embodiment of thepresent invention, the extract has antibacterial agent characteristics.

[0033] In yet another embodiment of the invention, the extract inhibitsgrowth of Gram-positive and Gram-negative bacteria.

[0034] In yet another embodiment of the invention, the yellow methanolicextract shows reversible colour change, being pink under alkaline andyellow under neutral or acidic conditions and is used as a pH indicator.

[0035] In another embodiment of the invention provides a composition fora sunscreen compound, said composition comprising, 25 to 75 mgmethanolic extract of the bacterium claimed in claim 1, with 4 to 8 mlglycerol, 1 to 3 ml polyxylene sorbitan monolaurate, 5 to 15 ml ethanoland water q.s, preferably contains 50 mg methanolic extract of thebacterium, with 6 ml glycerol, 2 ml polyxylene sorbitan monolaurate, 10ml ethanol and water q.s.

[0036] In yet another embodiment, the methanolic extract of novelbacterium is used for enhancing the color and shelf life of cheese oryoghurt.

[0037] In yet another embodiment, the amount of extract used forenhancing the color and shelf life of cheese and yoghurt ranging between0. 01 g/kg to 10 g/kg.

[0038] In yet another embodiment, the amount of extract used forenhancing the color and shelf life of cheese and yoghurt is 0.01 g/kg.

[0039] In another embodiment of the invention, dried methanolic extractis used for preparing menaquinone −7-8 containing substance, which areused in food and beverage and the quantity of extract used is in therange between 0.0001 to 0.001%.

[0040] Yet another embodiment provides a process for preparingmenaquinone −7,8 containing substance, said process comprising growingthe cells, for 2-5 days, harvesting after centrifugation and eitherspray drying or lyophilising and using at a concentration rangingbetween 0.5 to 10% for preventing and treatment of osteoporosis.

[0041] In yet another embodiment, the cells are grown for 4 days beforeharvesting.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0042] In the drawing accompanying this specification: FIG. 1 representsthe absorption spectrum of the extract.

[0043]FIG. 2 represents the absorption spectrum of methanolic extractpartitioned with petroleum ether.

[0044]FIG. 3 represents the IR spectra of crude methanolic extract.

[0045]FIG. 4 represents the NMR spectra of crude methanolic extract.

[0046] The following examples are given by way of illustration andtherefore should not be construed to limit the scope of the presentinvention .

EXAMPLE-1

[0047] Bacterial cells of the deep-sea isolate NIOCC isolate #222 wereused to prepare methanol extract. The cells were grown in standardnutrient broth (Himedia Bombay) in 100% sea water (pH 7.5) for 3 days ona7 rotary shaker at 120 rpm at 28±2° C. The cells were collected bycentrifugation at 5000 rpm and washed with 1.5% NaCl solution twice toremove the media. To the cells about 10 ml of 100% methanol was addedand extracted overnight at 5° C. The supernatant is collected aftercentrifugation at 10,000×g for 10 minutes. The pellet was againextracted twice with 1Oml and twice with 5-ml quantities of methanol.The extracts were pooled and the volume was made up to 30-ml. This wasconcentrated in a flash evaporator at 40° C. to 8 ml. The exampleillustrates the method for the preparation of methanol extract frombacterial cells.

EXAMPLE 2

[0048] The methanol extract prepared as shown in Example 1 was scannedfrom 260 nm to 550 nm and it showed a peak at 454 mn with shoulders at435 and 480 nm (FIG. 1). The same methanolic extract as shown in Example1 was partitioned with 10-ml petroleum ether and scanned from 260 to 550nm in a UV-visible spectrometer. In petroleum ether, peaks at 448 nmwith shoulders at 430 and 470 nm showed that it is a carotenold likecompound (FIG. 2). IR and NMR spectra of crude methanolic extracts aregiven in FIG. 3 and 4 respectively.

EXAMPLE 3

[0049] A small quantity (100 μl) of the extractas shown in Example 1 wasdiluted to 4 ml with 100% methanol and scanned from 280 to 400 nm. UVabsorbing property between 280 to 380 mn was observed with a peak at 340mn (UVA). This property can also be seen in (FIG. 1). Absence of claimsfor such properties in other species of allied bacteria is shown inTable 3.

EXAMPLE-4

[0050] Washed suspensions of cells of E. coli and the candidatebacterial isolate used in the present invention were exposed to brightsunlight between 1100 to 1200 hrs and aliquots were plated at every 15minute intervals. The current isolate could withstand the exposure andwas cultivable even after 45′ exposure to solar radiation having5.75mwcm⁻² at 312 nm (Table 1). TABLE 1 Survival of cultures whenexposed to sunlight. Time of exposure E. coli Candidate culture(minutes) CFU.M1⁻¹ CFU.M1⁻¹ 0 (control) 1.06 × 10⁸ 4.72 × 10⁷  (±0.02)*(±0.75) 15 1.72 × 10⁶ 5.41 × 10⁶ (±0.56) (±0.36) 30 3.36 × 10³ 2.34 ×10⁶ (±0.32) (±0.80) 45 0 7.68 × 10⁵ (±1.28)

[0051] CFU=Colony forming Units; Values in parenthesis show StdDeviation (SD)

EXAMPLE 5

[0052] The extract prepared as shown in Example 1 was tested forantibacterial properties against 2 Gram-positive (Bacillus subtilus andStreptococcus aureus) and 2 Gram-negative test cultures (Escherichia.Coli and Pseudomonas aeruginosa). Test cultures were grown ovemightin 5ml nutrient broth containing 0.8% agar to a density of 10¹² ml⁻¹ andpoured over nutrient agar plates to form a lawn.

[0053] 5 μl of methanolic extract prepared as shown in Example 1 wasspotted over sterile antibiotic discs of 6 mm dia (Himedia, Mumbai) intriplicates. One of these discs was alkalinised by adding 2 μl of 0.1NNaOH and the other acidified by adding 2 μl of 0.1N HCI. On each lawn 3discs in duplicate plates for each test culture were placed andincubated overnight at room temperature (28±2° C.). The zone ofinhibition was measured. Controls for methanol, acid and base were alsoincluded. While E. coli was inhibited in the acidic and neutral pH,Pseudomonas aeruginosa was inhibited in all three pHs. Bacillus subtiliswas inhibited in all three pHs but there was no effect on Staphylococcusaureus is shown in Table 2. Presence of such antagonistic properties inother species of bacteria is shown in table 3. TABLE 2 Antibioticactivity (inhibition zone in mms) of the extract of the candidatebacteria. Volume of Inhibition Zone extract E. coli P. aerugenosa S.aureus B. subtils 5 μl A B N A B N A B N A B N 6.2 0 6.2 6.4 6.7 6.4 0 00 6.4 6.7 6.4

EXAMPLE-6

[0054] About 100 μl of 1 N NaOH was added to 1 ml of dilute methanolextract (1:4). Colour turned from yellow to pink. The colour wasreversible with 1 N HCI. Thus it shows its pH indicator property.

EXAMPLE 7

[0055] A new cosmetic preparation contains methanolic extract of thebacteria 50 mg, Glycerol, 6.0ml, polyoxyethylene sorbitan monolaurate2.Oml, ethanol, 10.0 ml, purified water q.s. to make 100 ml.

EXAMPLE 8

[0056] Dried methanolic extracts as described in example 1 are added at0.01 g/kg of cheese or yoghurt to enhance colour and shelf life andfound enhance the color as well as the shelf-life of the cheese.

EXAMPLE 9

[0057] The culture is grown for 4 days and the cells harvested aftercontrifugation. The cells are either spray dried or lyophilised andincluded at 0.5-0.1% concentration for prevention and treatment ofosteoporosis. Dried methanolic extracts as described in example at0.001% could also be used alternatively.

[0058] The Main Advantages of the Present Invention are:

[0059] 1. The preparation could be used as a food additive that

[0060] a) augments colour;

[0061] b) improves shelf life by inhibiting both Gram-positive andGram-negative bacteria; and

[0062] c) indicates spoilage by colour change.

[0063] 2. The preparation could be used in skin cream/lotion not only asa radio protectant but also for preventing acne.

[0064] 3. As only one culture is used for all the properties, thenecessity to cultivate multiple organisms is eliminated. This isdefinitely a great advantage for commercial preparation. TABLE 3 Brevibacterium B spp 2, iodinum B. epidermis/ B. acetylicum Sl. No. Property 4,5, 6 B linens 1 2 B. casei 2 2 B. halotoleran 2 B maris 3 B.(NIO) 1Colour +/− + + − − − − + 2 antibacterial +/− + + + +/− − + + G +ve G +veand G −ve 3 UV − − − − − − − + absorbing compound 4 UV + − − − − − − − +Color 5 UV + − − − − − − − + antimicrobial 6 UV + − − − − − − − +Antimicrobial + − − − − − − − + Color

1. Novel deep-sea bacterium deposited with National Institute ofOceanography, Goa, India, having an accession no NIOCC isolate #222, andbeing deposited with International depository . . . having accession no. . . ,which has a similar properties to known Brevibacterium caseiisolated from the deep sea at 5000 m depth waters of the Indian Ocean.2. Novel bacterium as claimed in claim 1 is a baroduric (pressuretolerant) one i.e. it is capable of growing both at 500 atm and at 1 atmpressure.
 3. Novel bacterium as claimed in claim 1 wherein, thepetroleum ether fraction of the bacterium when scanned in an UV visiblespectrometer shows characteristic peaks at 448 nm with shoulders at 430and 470 nm, which is similar to the caratenoid compound.
 4. Novelbacterium as claimed in claim 1 wherein, the alcoholic extract of thesaid bacterium having caroteniods, UV absorption, anti bacterial, pHindicating properties.
 5. Novel bacterium as claimed in claim 1wherein,the extract of the bacterium is used in many industrial applications,such as a food and beverages additive and food additive colour cumpreservative.
 6. A process for the preparation of alcoholic extract ofdeep-sea bacterium isolated from the Indian coastal zones of Arabiansea, said process comprising isolating the bacterium and growing thecells in a medium with salinity ranging from 1.5 to 3% for 3-4 days at28±2° C., centrifuging and washing with 1.5% NaCl, extracting withalcohol for 2- 3 times and obtaining an extract which shows theproperties of carotenoids (yellow/orange colour), UV absorption,antibacterial and pH indicator.
 7. A process as claimed in claim 6wherein, the solvent used to extract is methanol.
 8. A process asclaimed in claim 6 wherein, the extract is used as UV (A, B, C)absorbing compound.
 9. A process as claimed in claim 6 wherein, theextract inhibits growth of Gram-positive and Gram-negative bacteria. 10.A process as claimed in claim 6 wherein, the yellow methanolic extractshows reversible colour change, being pink under alkaline and yellowunder neutral or acidic conditions and is used as a pH indicator.
 11. Acomposition for a sunscreen compound, said composition comprising, 25 to75 mg methanolic extract of the bacterium claimed in claim 1, with 4 to8 ml glycerol, 1 to 3 ml polyxylene sorbitan monolaurate, 5 to 15 mlethanol and water q.s.
 12. A composition as claimed in claim 11comprising 50 mg methanolic extract of the bacterium, with 6 mlglycerol, 2 ml polyxylene sorbitan monolaurate, 10 ml ethanol and waterq.s.
 13. Use of methanolic extract of novel bacterium for enhancing thecolor and shelf life of cheese or yoghurt.
 14. Use of methanolic extractas claimed in claim 13 wherein, the amount of extract used for enhancingthe color and shelf life of cheese and yoghurt ranging between 0.01 g/kgto 10 g/kg.
 15. Use of methanolic extract as claimed in claim 13wherein, the amount of extract used for enhancing the color and shelflife of cheese and yoghurt is 0.01 g/kg.
 16. Use of dried methanolicextract for preparation of menaquinone −7-8 containing substance for usein food and beverage.
 17. Use as claimed in claim 16 wherein, thequantity of extract used in the range between 0.0001 to 0.001%.
 18. Aprocess for preparing menaquinone −7,8 containing substance, saidprocess comprising growing the cells claimed in claim 1, for 2-5 days,harvesting after centrifugation and either spray drying or lyophilisingand using at a concentration ranging between 0.5 to 10% for preventingand treatment of osteoporosis.
 19. A process as claimed in claim 18wherein, growing the cells for 4 days before harvesting.